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D Renz

Publications and source records attributed to D Renz.

At least 19 recordsLinked to original sources
PubMed2002

Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1.

In vivo, hypoxia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Primary cultures of porcine brain derived microvascular endothelial cells were used as an in vitro BBB model to evaluate the mechanisms by which hypoxia regulates paracellular permeability. Paracellular passage across endothelial cell monolayers is regulated by specialized intercellular structures like the tight junctions (TJ). Zonula occludens-1 (ZO-1), a protein of the TJ, lines the cytoplasmic face of intact TJ. The continuity of the ZO-1 expression was disrupted during 24 h of hypoxia which correlated with a decrease of the protein level to 32 +/- 8% and with a twofold increase in the phosphorylation of ZO-1 in comparison to values determined at the start of the experiment. The localization and expression level of ZO-1 were maintained during hypoxia in the presence of a polyclonal antibody to vascular endothelial growth factor (VEGF) demonstrating that hypoxia-induced changes of the ZO-1 expression are mediated by VEGF. The effect of hypoxia on the ZO-1 distribution probably is not tissue- or cell-specific because similar changes of ZO-1 distribution were observed when the rat brain endothelial cell line RBE4 or the murine epithelial cell line CSG was used. Furthermore, ZO-1 changes correlated with small changes in actin distribution. These results suggest that hypoxia increases the paracellular flux across the cell monolayer via the release of VEGF, which in turn leads to the dislocalization, decreased expression, and enhanced phosphorylation of ZO-1. Science.

Actins↗
PubMed2001

In vitro effects of dexamethasone on hypoxia-induced hyperpermeability and expression of vascular endothelial growth factor.

Clinically, dexamethasone is known to reduce cerebral edema. To further investigate the mechanism of this neuroprotection, an in vitro model of brain-derived microvessel endothelial cells (BME cells) was used to investigate the effect of dexamethasone on hypoxia-induced hyperpermeability. Furthermore, the expression of vascular endothelial growth factor (VEGF), which is known to be the mediator of hypoxia-induced hyperpermeability, was evaluated. Dexamethasone (40 microg/ml=100 microM) decreased hypoxia-induced permeability and VEGF expression significantly during time periods of more than 3 h. The time dependence of the dexamethasone effect correlated with a changed mechanism by which hypoxia induced VEGF expression. This was deduced because hypoxia-induced hyperpermeability and VEGF mRNA level were decreased in the presence of an antisense oligonucleotide coding for a region which binds a mRNA stabilizing protein, but only up to 3 h of hypoxia. Furthermore, during this time period the half-life of VEGF mRNA was increased. Results suggest that dexamethasone only decreases transcriptional-induced VEGF expression and that this may be related to the efficacy of dexamethasone to treat brain edema.

Animals↗
PubMed2000

Effect of astroglial cells on hypoxia-induced permeability in PBMEC cells.

An in vitro model of the blood-brain barrier (BBB), consisting of porcine brain-derived microvascular endothelial cells (PBMEC), was used to evaluate the effect of astrocytes in the BBB disruption during hypoxia. Hypoxia-induced hyperpermeability was decreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, when glia cell-conditioned medium was used. Corresponding to effects on hypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor (VEGF) mRNA and protein expression, which recently was shown to be responsible for hypoxia-induced permeability changes in vitro. The effect on hypoxia-induced hyperpermeability and VEGF expression was specific for astroglia cells because conditioned medium from bovine smooth muscle cells (BSMC) did not show any effect. Immunocytochemistry revealed that 24 h of hypoxia disrupted the continuity of the tight junction protein, zonula occludens-1 (ZO-1), which lines the cytoplasmic face of intact tight junctions. These changes were prevented when hypoxia was performed in glia cell-conditioned medium. Results suggest that astrocytes protect the BBB from hypoxia-induced paracellular permeability changes by decreasing hypoxia-induced VEGF expression in microvascular endothelial cells.

Animals↗
PubMed1999

Hypothermia abolishes hypoxia-induced hyperpermeability in brain microvessel endothelial cells.

The effect of mild (32 degrees C) and deep (22 degrees C) hypothermia on hypoxia-induced hyperpermeability was examined using an in vitro model of brain derived microvascular endothelial cells (BMEC). It was shown that hypoxia-induced hyperpermeability to inulin across the BMEC monolayer was completely abolished at 32 degrees C and 22 degrees C for up to 24 h of hypoxia. During normoxia, no influence of hypothermia on BMEC monolayer permeability was observed. The hypoxia-induced decrease of the cyclic AMP level after 6 h was abolished at 32 degrees C as well as at 22 degrees C of hypoxia. But after 24 h of hypoxia, hypothermia did no longer prevent the hypoxia-induced decrease of the cAMP level, which suggests that the effect of hypothermia on hypoxia-induced hyperpermeability is not caused by maintenance of the cAMP level. Because vascular endothelial growth factor (VEGF) has been shown to be the mediator of hypoxia-induced permeability changes of BMEC via the release of nitric oxide (NO), the effect of hypothermia on the VEGF expression was evaluated. During normoxia, hypothermia did not change the VEGF expression significantly but the hypoxia-induced increase in VEGF mRNA and protein expression was completely abolished at 32 degrees C and 22 degrees C respectively. Accordingly, the hypoxia-induced increase of the cGMP level was depressed by hypothermia, which demonstrates that also the amount of NO released during hypoxia is decreased at lower temperatures. Results suggest that deep as well as mild hypothermia decreased hypoxia-induced hyperpermeability by lowering the expression of the permeability-increasing protein VEGF and with it the release of NO.

Adenosine Triphosphate↗
PubMed1999

Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO.

In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.

Animals↗
PubMed1998

Barbiturates decrease the expression of vascular endothelial growth factor in hypoxic cultures of porcine brain derived microvascular endothelial cells.

Vascular endothelial growth factor (VEGF) is known to be produced in higher amounts during hypoxia by a variety of cell types and has been shown to increase the permeability of brain derived microvascular endothelial cells (BMEC) during hypoxia by an autocrine mechanism. Because the barbiturates, methohexital (MH) and thiopental (TP), induced a dose-dependent reduction in hypoxia-induced permeability changes of BMEC, the effect of both barbiturates on the VEGF expression during hypoxia was investigated. Both barbiturates decreased the hypoxia-induced expression of VEGF in BMEC in a concentration-dependent manner. This effect is partly caused by the impairment of the hypoxia-induced VEGF mRNA stabilization. VEGF-induced permeability changes during normoxia were unaffected by the barbiturates suggesting that MH and TP are directly reducing hypoxia-induced VEGF synthesis. In conclusion, the inhibiting effect of these barbiturates on the hypoxia-induced VEGF expression results in the decreased permeability of the BMEC monolayer during hypoxia, which may contribute to the described neuroprotective action of barbiturates by reduction of brain edema formation.

Adenosine↗
PubMed1998

On the fate of orally ingested foreign DNA in mice: chromosomal association and placental transmission to the fetus.

We have previously shown that, when administered orally to mice, bacteriophage M13 DNA, as a paradigm foreign DNA without homology to the mouse genome, can persist in fragmented form in the gastrointestinal tract, penetrate the intestinal wall, and reach the nuclei of leukocytes, spleen and liver cells. Similar results were obtained when a plasmid containing the gene for the green fluorescent protein (pEGFP-C1) was fed to mice. In spleen, the foreign DNA was detected in covalent linkage to DNA with a high degree of homology to mouse genes, perhaps pseudogenes, or to authentic E. coli DNA. We have now extended these studies to the offspring of mice that were fed regularly during pregnancy with a daily dose of 50 microg of M13 or pEGFP-C1 DNA. Using the polymerase chain reaction (PCR) or the fluorescent in situ hybridization (FISH) method, foreign DNA, orally ingested by pregnant mice, can be discovered in various organs of fetuses and of newborn animals. The M13 DNA fragments have a length of about 830 bp. In various organs of the mouse fetus, clusters of cells contain foreign DNA as revealed by FISH. The foreign DNA is invariably located in the nuclei. We have never found all cells of the fetus to be transgenic for the foreign DNA. This distribution pattern argues for a transplacental pathway rather than for germline transmission which might be expected only after long-time feeding regimens. In rare cells of three different fetuses, whose mothers have been fed with M 13 DNA during gestation, the foreign DNA was detected by FISH in association with both chromatids. Is maternally ingested foreign DNA a potential mutagen for the developing fetus?

Administration, Oral↗
PubMed1997

Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA.

Food-ingested foreign DNA is not completely degraded in the gastrointestinal tract of mice. Phage M13mp18 DNA as a test molecule devoid of homology to mouse DNA was pipette-fed to or added to the food supply of mice. The fate of this foreign DNA in the animals was followed by several methods. In 84 animals, fragments of M13mp18 DNA were detected in the contents of the small intestine, the cecum (until 18 h), the large intestine, or the feces. In 254 animals, M13mp18 DNA fragments of up to 976 bp were found in blood 2-8 h after feeding. In buffer-fed control animals, M13mp18 DNA could not be detected. M13mp18 DNA fragments were traced by PCR in peripheral leukocytes and located by fluorescent in situ hybridization in about 1 of 1000 white cells between 2 and 8 h, and in spleen or liver cells up to 24 h after feeding, but not later. M13mp18 DNA could be traced by fluorescent in situ hybridization in the columnar epithelial cells, in the leukocytes in Peyer's patches of the cecum wall, in liver cells, and in B cells, T cells, and macrophages from spleen. These findings suggest transport of foreign DNA through the intestinal wall and Peyer's patches to peripheral blood leukocytes and into several organs. Upon extended feeding, M13mp18 DNA could be recloned from total spleen DNA into a lambda vector. Among about 2.5 x 10(7) lambda plaques, one plaque was isolated that contained a 1299 nucleotide pair fragment (nt 4736-6034) of sequence-identified M13mp18 DNA. This fragment was covalently linked to an 80 nt DNA segment with 70% homology to the mouse IgE receptor gene. The DNA from another lambda plaque also contained mouse DNA, bacterial DNA, and rearranged lambda DNA. Two additional plaques contained M13mp18 DNA fragments of at least 641 (nt 2660-3300) or 794 (nt 4640-5433) nucleotide pairs. The medical and evolutionary implications of these observations may be considerable.

Animals↗
PubMed1997

Role of adenosine in the hypoxic induction of vascular endothelial growth factor in porcine brain derived microvascular endothelial cells.

Hypoxia induced the mRNA expression of vascular endothelial growth factor (VEGF) in porcine brain derived microvascular endothelial cells (BMEC) in a time-dependent manner. Corresponding to the mRNA induction the protein level of VEGF was elevated during hypoxia. The adenosine A1 receptor antagonist 8-phenyltheophylline (8-PT) reduced the hypoxia-induced VEGF mRNA and protein expression significantly. The treatment of BMEC with cobalt chloride-known to activate an oxygen sensing mechanism similar to the one used by the erythropoietin gene-also induced the VEGF mRNA expression, but 8-PT did not reduce this VEGF induction. Although, earlier studies revealed that agents like phorbolester induced the VEGF mRNA expression, the specific inhibitor of the proteinkinase C (PKC) bisindolylmaleimide (BIM) did not reduce but enhanced the hypoxia-induced VEGF mRNA expression. These results indicate that the VEGF induction in BMEC can proceed through PKC-dependent and -independent pathways (like those acting via the putative oxygen sensor). Hypoxia in BMEC probably activates the PKC-dependent pathway mainly via adenosine which might be formed during hypoxia and thereby inhibits activation of PKC-independent, oxygen sensing, pathways. This suggestion was supported by the fact that hypoxia as well as adenosine increased the VEGF mRNA expression post-transcriptionally by enhancing the stability of the VEGF mRNA [corrected].

Adenosine↗
PubMed1996

DNA methylation in the promoter of ribosomal RNA genes in human cells as determined by genomic sequencing.

Many RNA polymerase II- or III-transcribed genes are inactive when their promoter is methylated at critical CpG dinucleotides. We have applied the genomic sequencing method and a direct DNA blotting technique to analyze the extent of DNA methylation in the 5'-CpG-3' rich promoter region of the RNA polymerase I-transcribed ribosomal RNA genes (rDNA) in DNA from primary human cells, primary human tumor cells and human cell lines. In none of the analyzed primary human cells and primary human tumor cells was the DNA in the rDNA promoter region found to be detectably methylated. In contrast, in some of the cell lines this promoter is methylated in all 5'-CpG-3' dinucleotides in the majority of the approximately 200 ribosomal RNA gene copies. In actively growing cells, rDNA gene activity is a prerequisite for cell viability. The high levels of DNA methylation in the promoter region of rDNA in the human cell lines raise questions on the role of promoter methylation in these RNA polymerase I-transcribed genes. It is, however, conceivable that a subset of the about 200 rDNA copies per haploid genome have escaped methylation and account for the rRNA synthesis in these cell lines. Alternatively, complete 5'-CpG-3' promoter methylation may be compatible with promoter activity as demonstrated for certain viral genomes.

Base Sequence↗
PubMed1996

Effects of barbiturates on hypoxic cultures of brain derived microvascular endothelial cells.

An in vitro model of the blood-brain barrier (BBB) consisting of porcine brain derived microvascular endothelial cells (BMEC) seeded onto collagen-coated polycarbonate membranes was used to investigate the effects of the barbiturates, methohexital and thiopental, on permeability properties of the endothelial cell monolayer under hypoxia. The permeability of cultured BMEC to ions and sucrose increased significantly during 6 h of hypoxia in a reversible manner. Cells were resistant to hypoxia for up to 24 h, but 48 h resulted in marked damage as assessed by the release of lactate dehydrogenase activity into the culture medium. The hypoxia-induced increase of the permeability was unchanged in the presence of superoxide dismutase (SOD) and catalase. Methohexital and thiopental decreased the hypoxia-induced permeability increase in a concentration-dependent manner and permeability changes were abolished completely at the barbiturate concentration of 50 micrograms/ml. The barbiturates had no effect on the intracellular cAMP content which started to decline after 3 h of hypoxia. Results suggest that barbiturates at high concentrations might be able to prevent permeability changes of the BBB during cerebral ischemia.

Animals↗
PubMed1995

Transcriptional silencing of human Alu sequences and inhibition of protein binding in the box B regulatory elements by 5'-CG-3' methylation.

In earlier work, we demonstrated that 5'-CG-3' methylation inhibits the transcriptional activity of human Alu elements associated with the alpha 1-globin and the angiogenin genes in a cell-free transcription system from HeLa nuclear extracts. These studies have been extended to different Alu sequences and to investigations on the mechanism involved in transcriptional silencing by methylation. By comparing the results of DNase I and dimethyl sulfate (DMS) in vitro footprinting on a consensus sequence in the RNA polymerase III promoter control B region between the unmethylated and the 5'-CG-3' methylated B box, evidence has been adduced for effects of 5'-CG-3' methylation on the interaction of specific nuclear proteins with DNA sequences in the B control region of the Alu elements. These results are consistent with the interpretation that the 5'-CG-3' methylation interferes with the binding of proteins that are essential for the function of the B control region in these RNA polymerase III-transcribed elements, and that promoter methylation thus inhibits transcription.

Base Sequence↗
PubMed1995

In vitro effects of fentanyl, methohexital, and thiopental on brain endothelial permeability.

BACKGROUND: The use of anesthetics can lead to changes of the permeability of the blood-brain barrier (BBB). To eliminate those factors, such as varying hemodynamic effects that are associated with anesthesia, an in vitro model of the BBB consisting of brain microvascular endothelial cells (BMEC) was used to study the direct effects of the opiate, fentanyl, and the barbiturates methohexital and thiopental, which are widely used in the clinical setting, on the permeability of confluent monolayers. METHODS: BMEC isolated from porcine brains were grown to confluence on collagen-coated polycarbonate membranes, which were placed into 24 well dishes, thus forming a two-compartment chamber. The permeability of the BMEC monolayer to ions--determined by measurements of the transendothelial resistance (TER)--the passage of sucrose, Evans Blue albumin (EBA), and alpha-aminoisobutyric acid (AIB) across the BMEC monolayer were assessed in the presence and absence of fentanyl (25-100 ng/ml), methohexital (10-50 micrograms/ml), and thiopental (25-100 micrograms/ml). RESULTS: The permeability of cultured BMEC to the tracers used increased significantly after exposure of the monolayer to arabinose and after removal of calcium ions. Fentanyl, methohexital, and thiopental did not change the permeability of the cell monolayer to ions, sucrose, albumin, and AIB. Only thiopental at the concentration of 100 micrograms/ml increased the flux of AIB. CONCLUSIONS: At the concentrations tested, there is little evidence of changes in the permeability of the in vitro BBB caused by fentanyl, methohexital, and thiopental regarding the para- and transcellular route of ions, sucrose, and albumin. Only thiopental at a concentration of 100 micrograms/ml increased the passage of AIB across the BMEC monolayer.

Albumins↗
PubMed1994

Variability in allelic DNA methylation in spermatozoa.

In certain segments of human DNA, the methylation of deoxycytidine residues has been found to be highly specific and interindividually conserved. Imprinted DNA sequences in diploid primary cells show allele-specific differences in DNA methylation, usually with the active chromosomal regions being unmethylated and the inactive regions being methylated. We show here that DNA from spermatozoa exhibits variations in allelic methylation patterns. Since germ cells are haploid, individual spermatozoa can differ in DNA methylation patterns not only in the maternally or paternally derived allele, but also within each allele.

Alleles↗